For years, a major goal of tumor immunology has been the molecular characterization of human tumor antigens. Missense mutations in the p53 tumor suppressor gene are the most common genetic events associated with human cancer. Our work has focused on an immunological characterization of p53 missense mutations occurring in cytotoxic T-cell defined p53 epitopes in HLA-A0201+ squamous cell carcinomas of the head and neck. An analysis of p53 expressed in these tumors identified six missense mutations that occurred within the three HLA-A2-restricted, CTL-defined p53149-157, p53217-225 and p53264-272 epitopes. 3/6 mutations yielded mutant peptides with reduced or similar binding affinities for HLA-A2 molecules compared to the parental peptides, which failed to yield anti-peptide CTL following in vitro stimulation (IVS) of peripheral blood mononuclear cells (PBMC) with peptide-pulsed autologous dendritic cells. Two p53 mutations, which occur within the p53217-225 epitope, Y220C and Y220H, generated mutant peptides with slightly increased affinities to the HLA-A 0201 molecule relative to the weakly binding parental peptide, that were immunogenic. The anti-p53 Y220C effectors were reactive against an HLA-A0201+ SCCHN cell line, UD-SCC 6, which expresses the p53 Y220C mutation, indicating that this mutation can be naturally presented. IVS of the UD-SCC 6 patients' PBMC generated an anti-p53 Y220C CTL that recognized the autologous tumor cell line. Most importantly, the Y220C mutation was detected in 4/40 (10%) SCCHN in HLA-A2+ patients compared to a reported occurrence frequency of 1% of all human cancers. The observed increased representation of mutations at p53 codon 220 in HLA-A2.1+ SCCHN tumors, which also might be prevalent in other HLA-A2.1+ carcinomas, suggests that development of vaccines targeting this mutation would be more widely applicable than previously envisioned for any given p53 missense mutation. T cell activation is a critical step in mounting an appropriate immune defense against infectious agents such as viruses and bacteria. Central to understanding how antigen presenting cells mediate the full activation of T cells is knowledge of the interaction of MHC molecules with the T-cell receptor (TCR). We have previously described a novel experimental system in order to address how engagement of TCR leads to full T cell activation. We have isolated the mouse AHIII TCR and its MHC ligand, H2-Db-p1058, as well as a human pMHC complex with which it interacts, HLA-A2-p1049. Having demonstrated that we have in possession a functional AHIII cytotoxic T cell line and can produce large quantities of active protein for use in functional and biochemical analysis, we are currently performing experiments to distinguish between two models of T cell activation, the affinity-based model and the kinetic-rate model. Using our co-crystal structure of AHIII and HLA-A2-p1049, we have selected 11 mutations in the A2-p1049 molecule that will alter the affinity and/or kinetic off rate for the AHIII TCR. In the past year we have finished making sufficient quantities of the mutant pMHC complexes to determine its affinity and kinetic binding rates to the AHIII TCR by surface plasmon resonance. To relate these mathematical constants to T cell activity, we previously performed cytotoxic lysis assays. Realizing that cellular proliferation and cytokine secretion also make up the biological outcome of full T cell activation, we sought to establish the tools necessary to measure these responses. Thus, in the past year, we have finished making the cell lines expressing our mutant pMHC complexes needed to elicit T cell proliferation and cytokine secretion. Additionally, we have now obtained co-crystals of the AHIII TCR and some mutant pMHC complexes that will aid us in understanding how these mutations have affected the kinetic rate constants. Our current data supports the affinity-based model. The current study focuses on altered stimuli on the T cell and examines distal functional consequences, names cytotoxic lysis activity, proliferative response, and cytokine secretion. Upon the completion of this study in the near future, it will be essential to examine more immediate responses, such as changes in gene expression, activation of signaling intermediates, and spatial rearrangement of signaling components, in order to understand how the distal functional consequences are achieved. The establishment of an AHIII transgenic mouse will be necessary to compare our results with true primary T cells. Additionally, an AHIII transgenic mouse will allow this same study to be performed on naive and memory T cells, as opposed to cytotoxic T cells, in order to examine the differences in the requirements for activation.